The long term objective of the proposed research is to determine the role of cofactor availability in the regulation of gene expression. The specific aims are to use in vivo and in vitro models of riboflavin deficiency to study the role of that vitamin in regulating the expression of the short-chain acyl-CoA dehydrogenase (SCAD) gene. SCAD is an FAD dependent dehydrogenase of the mitochondrial beta-oxidation pathway. In young rats, this enzyme undergoes a characteristic post weaning maturation, during which its activity increases by approximately 4-fold over a 3-4 day period. Riboflavin deficiency blocks this development. To examine further the mechanisms by which riboflavin availability regulates the activity of the enzyme, a hepatoma cell culture system of riboflavin deficiency was developed in which treatment with riboflavin causes a 50-100 fold increase in SCAD activity. Preliminary data suggests that the increased SCAD activity reflects increased enzyme mass. The specific aims of the proposed project are to 1) determine the level at which SCAD gene expression is influenced by riboflavin availability and 2) characterize the SCAD gene sequences that mediate the response to either riboflavin or a riboflavin-derived factor. These aims will be approached by isolating a cDNA clone encoding SCAD and using it as a probe to measure SCAD mRNA levels during the developmental period in weanling rats, to compare the levels in riboflavin-deficient and normally fed rats and to assess changes in mRNA level in cell culture when riboflavin-deficient cells are treated with the vitamin. The cDNA clone will then be used to estimate SCAD genomic clones. The long range goal of the research is to identify gene sequences that may comprise a riboflavin-response element. This will be approached by subcloning putative regulatory sequences into a vector containing the chloramphenicol acetyl transferase structural gene, transfecting the vector into the cultured cells and examining the expression of CAT in response to riboflavin treatment.